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The Sir2 family of protein deacetylases
The Sir2 family of protein deacetylases John M Denu The importance of NAD+-dependent deacetylases (Sir2 family or sirtuins) in cell survival, ageing and apoptosis has ignited a flurry of both chemical and cellular investigations aimed at understanding this unique class of enzymes. This review focuses on recent mechanistic advances that highlight structure, catalysis, substrate recognition and interactions with small-molecule effectors. Recent X-ray structures revealed binding sites for both NAD+ and acetyl-peptide. Biochemical studies support a two-step chemical mechanism involving the initial formation of a 10 -O-alkylamidate adduct formed between the acetyl-group and the nicotinamide ribose of NAD+. Acetyl transfer to the 20 ribose and addition of water yield deacetylated peptide and 20 -O-acetyl-ADP-ribose, a potential second messenger. Also, the molecular basis of nicotinamide inhibition was revealed, and sirtuin activators (resveratrol) and inhibitors (sirtinol and splitomicin) were identified through small-molecule library screening. Addresses Department of Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706, USA Corresponding author: Denu, John M ([email protected])
Current Opinion in Chemical Biology 2005, 9:431–440 This review comes from a themed issue on Mechanisms Edited by Rowena G Matthews and Christopher T Walsh Available online 24th August 2005 1367-5931/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2005.08.010
Introduction The NAD+-dependent protein deacetylases have emerged as important regulators of diverse biological processes [1,2]. These enzymes, referred to as sirtuins or Sir2-like proteins, constitute the class III histone deacetylases (HDACs) and are conserved from bacteria to humans [3,4]. The founding member yeast Sir2 (ySir2, yeast silent information regulator 2) is essential for maintaining silent chromatin through the deacetylation of histones (Figure 1a) [5,6]. Sir2 enzymes have also been implicated in mediating lifespan increases in yeast, worms and flies [7–9]. Additional reports over the past several years indicate a much broader range of substrates and functions for sirtuins [1,10,11]. For example, short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast are regulated by reversible acetylation, with www.sciencedirect.com
sirtuins functioning as activating deacetylases [12,13]. Among the most studied human Sir2 homologs, SIRT1 was shown to regulate non-histone substrates including p53 [14–16], FOXO proteins [17–19], p300 [20], NFkB [21] and PGC-1a [22,23] (Figure 1b), implicating sirtuins in apoptosis, cell survival, transcription and metabolism. The role of sirtuins in controlling basic cellular functions, particularly apoptosis and cell survival, has spurred great interest in understanding the molecular mechanisms of catalysis, substrate specificity and regulation, as well as in the development of small-molecule regulators that either activate or inhibit their cellular function. Pharmaceuticals that target sirtuins may be useful in the treatment of ageing, cancer, diabetes and neurodegeneration. Here, recent progress in our knowledge of catalysis, structure, regulation, substrate specificity and small-molecule effectors is presented.
Deacetylation reaction and biological implications In cases where clear enzymatic activity has been measured, sirtuins catalyze NAD+-dependent e–N-acetyl-lysine deacetylation from histones and non-histone proteins [24–28]. The deacetylase reaction requires NAD+ and produces deacetylated protein, nicotinamide and the unique metabolite O-acetyl-ADP-ribose (OAADPr) (Figure 1b) [26,27,29]. Sirtuins transfer the acetyl group from proteins and peptides to the 20 -OH of the nicotinamide ribose, yielding 20 -O-acetyl-ADP-ribose (20 -OAADPr) [29–31]. The nicotinamide ribosyl bond is cleaved and one net water molecule is added to the nicotinamide ribose. This reaction is in stark contrast to class I and II histone/protein deacetylases, which utilize an active site zinc, produce acetate, and do not require NAD+ [32]. The uniqueness of the sirtuin reaction begs many fundamental questions concerning the catalytic mechanism and its biological relevance. Why is NAD+ consumption required for a deacetylation reaction that is already thermodynamically favored? And, why is the acetyl group specifically transferred not to water but to the ADP-ribose portion of NAD+ to generate OAADPr? How does the enzyme accomplish this novel chemical reaction? Coupling deacetylation to NAD+ would be one mechanism to link changes in cellular NAD+ levels with deacetylation activity. Although a strong correlation between total NAD+ levels in cells and Sir2 function remains to be established, several genetic studies have provided ample evidence for a link between enzymes involved in NAD+ biosynthesis and sirtuin function [33,34,35,36–38]. It may be that localized synthesis of NAD+ is directly responsible for enhanced ySir2 and SIRT1 activity in these studies. Another explanation for Current Opinion in Chemical Biology 2005, 9:431–440
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Figure 1
Proposed functions of yeast Sir2 and of mammalian SIRT1. (a) Yeast Sir2 histone deacetylation and silencing function. Acetyl-CoA-dependent histone acetyltransferases acetylate histones leading to transcriptional activation. The NAD+-dependent deacetylase Sir2 deacetylates histones and creates transcriptional silent chromatin. Nicotinamide is a product of the reaction and is a potent inhibitor of Sir2. OAADPr is a novel metabolite generated during deacetylation and may function in linked cellular pathways. An NAD+ salvage pathway depletes nicotinamide and funnels NAD+ to Sir2. (b) General chemical reaction catalyzed by sirtuins. Human SIRT1 is depicted with several reported acetylated protein targets and the pathways affected.
the NAD+ dependence is that protein deacetylation is designed to create OAADPr, a proposed second messenger [39]. Microinjection of OAADPr was shown to inhibit oocyte maturation and to block cell division in starfish blastomeres [40]. Unidentified enzymes found in several species including human cells are able to efficiently metabolize OAADPr in extracts [40,41], suggesting that cellular pathways exist to tightly control OAADPr levels. Thus, OAADPr may act as a secondary messenger, a cofactor, or as a metabolic intermediate that couples deacetylation of target proteins to other cellular pathways [39]. Further studies are needed to uncover the functions of OAADPr, particularly its role in known processes regulated by sirtuins. Current Opinion in Chemical Biology 2005, 9:431–440
Reaction mechanism Recent kinetic studies indicate that sirtuins first bind acetylated substrate followed by NAD+ to form a ternary enzyme complex [42]. Conformational rearrangement of the NAD+ binding pocket upon acetyl-peptide binding was observed in the X-ray structure of a binary complex between archaeal Sir2 enzyme, Sir2-Af2, and an acetylated p53 peptide [43], providing additional evidence for ordered substrate binding. Although binary complexes of enzyme with NAD+ or ADP-ribose were reported initially [31,44], the nicotinamide moiety was not observed until recently, when the position of the nicotinamide base was pinpointed within the highly conserved C-pocket [45,46,47], a buried depression previously www.sciencedirect.com
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Figure 2
Proposed catalytic mechanism for the sir2 family of deacetylases.
noted near the predicted active site and implicated in catalysis [44]. As proposed in Figure 2, the initial chemical step involves cleavage of the nicotinamide-ribosyl bond of NAD+ and the concomitant attack of the carbonyl oxygen of an acetyl group from bound peptide to form a 10 -O-alkylamidate adduct between the two substrates (reviewed in [39,48]). Nicotinamide is released and subsequent activation of the ribose 20 -OH leads to nucleophilic attack at the iminium intermediate and generation of a 10 -20 -cyclic species. Water attack on the cyclic intermediate and general-acid catalyzed elimination of the e-amine of lysine yields the final two products 20 -OAADPr and deacetylated peptide, which are released in random fashion [42]. Evidence for a two-step chemical mechanism comes from the observation of enzyme-catalyzed nicotinamide:NAD+ www.sciencedirect.com
exchange (transglycosidation) [24,49,50], from rapidquench flow studies [26,42] and from the use of 20 deoxy-20 -fluoro-NAD+ as a surrogate co-substrate [49]. In the presence of acetyl-peptide and NAD+, exogenously added [14C]-nicotinamide is quickly incorporated into [14C]-NAD+ during the nicotinamide:NAD+ exchange reaction. This rapid transglycosidation with free nicotinamide to regenerate b-NAD+ is proposed to occur by the facile nucleophilic attack of nicotinamide on the putative 10 -O-alkylamidate intermediate [49,50]. Consistent with the formation of a 10 -O-alkylamidate adduct during deacetylation, replacement of NAD+ with 20 -deoxy-20 fluoro-NAD+ halted the deacetylation reaction but not the nicotinamide exchange reaction [49]. Rapid quenching studies with HST2 demonstrated that the nicotinamide–ribosyl bond is cleaved to form the putative 10 -O-alkylamidate intermediate at a rate of 8 s1, while the rate of subsequent acetyl-transfer to the 20 -OH ribose Current Opinion in Chemical Biology 2005, 9:431–440
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occurred at a rate of 2 s1. The kcat is 0.2 s1, suggesting that a product release step may limit turnover [42]. Sirtuin deacetylation reactions carried out in 18OH2, incorporate one 18O atom into the acetyl group of product 20 -OAADPr ([29]; JM Denu, BC Smith, unpublished data). Recently, the fate of the carbonyl oxygen of the acetylated peptide was determined using an 18O-label in the acetyl group of substrate (JM Denu, BC Smith, unpublished data). These recent studies demonstrated that the carbonyl oxygen of substrate is enzymatically transferred to the 10 position of product 20 -OAADPr. These new data provide the first direct evidence that the acetyl-group carbonyl oxygen initially attacks at the 10 position to yield the proposed 10 -O-alkylamidate intermediate (Figure 2). In the last chemical step, solvent isotope exchange data ([29]; JM Denu, BC Smith, unpublished data) are consistent with a water molecule attacking the 10 -20 -cyclic intermediate, incorporating one solvent molecule into the acetyl group of 20 -OAADPr. Crystal data have shown that the side chain of an invariant histidine residue is hydrogen bonded to the 30 -OH of the nicotinamide ribose and is suggested to act as a general base by initiating the deprotonation of the 20 -OH (acting through the 30 -OH) for attack on the 10 -O-alkylamidate intermediate, forming the 10 -20 -cyclic intermediate (Figure 2). This same histidine residue may serve as a general acid in the last step of catalysis, protonating the lysine leaving group (Figure 2).
Adp-ribosylation In several reports, sirtuins were suggested to harbor NAD+-dependent ADP-ribosyltransferase activity, transferring ADP-ribose to a protein acceptor or to the enzyme itself [3,28,51–53]. Weak or nonexistent deacetylation activity from some sirtuins has fueled this notion. However, there is currently insufficient evidence to unequivocally assign bona fide protein ADP-ribosyltransferase activity. Although sirtuins acting as ADP-ribosyltransferases remains a distinct possibility, the ability of sirtuins to catalyze multiple rounds of protein ADP-ribosylation has not been demonstrated. Among the reported cases, there are three possible mechanisms for the observed ADP-ribosylation (Figure 3). The first possibility is that some sirtuins indeed do possess ADP-ribosyltransferase activity, transferring the ADP-ribose of NAD+ to an acceptor protein (Figure 3a). In this scenario, an acetylated substrate is not required. In a second possibility, an acetylated substrate and NAD+ would be required to initiate the formation of the O-alkylamidate intermediate, which may be susceptible to directed or non-specific nucleophilic attack by the acceptor protein (Figure 3b), which may include acetylated protein itself. This mechanism is similar to the reaction (transglycosidation) of nicotinamide with the enzyme intermediate. After collapse of the intermediate to form the ADP-ribosylated Current Opinion in Chemical Biology 2005, 9:431–440
product, the acetylated residue would be regenerated. A third model requires NAD+ and acetylated substrate to produce OAADPr or its hydrolyzed product ADP-ribose (Figure 3c). Both OAADPr and ADP-ribose can react nonenzymatically with proteins to yield the ADP-ribosylated protein products (T Kowieski, JM Denu, unpublished data). The observation of sirtuin-dependent ADP-ribosylation with hemi-acetylated histone preparations should be analyzed with caution, as the resulting OAADPr produced by deacetylation can react with resident proteins non-enzymatically. In vivo, the possibility that OAADPr could serve as a substrate for a novel ADPribosyltransferase should be considered as well.
Structure and substrate specificity Recent X-ray structures of liganded sirtuin complexes have defined the binding pockets for both substrates and have provided supporting evidence for the mechanism proposed in Figure 2. The catalytic core of sirtuins consists of an NAD+-binding domain that is a variant of the Rossmann fold and a smaller subdomain composed of a helical module and a zinc-binding module (Figure 4). A large groove at the interface creates the active site where NAD+ binds. Acetylated peptide binds along an enzyme cleft and forms an enzyme-substrate b sheet with two flanking strands from the enzyme [43]. The acetyl-lysine side chain protrudes into a hydrophobic tunnel that terminates near the nicotinamide ribose of NAD+ [46,47]. Modeling studies based on the binary complexes of Sir2Af2 with NAD+ [46] and of Sir2Af2 with an acetylated-lysine peptide [43] suggest that the acetyl-group would be positioned on the a-face of the nicotinamide ribose of the catalytically competent ternary complex (Figure 4). Zhao et al. reported the ternary structure of HST2 bound to both a non-hydrolyzable NAD+ analog and an acetylate histone peptide [47]. These authors observed the acetyl group hydrogenbonding to the ribose 20 and 30 OH of the NAD+ analog. In this orientation, the carbonyl oxygen of the acetyl-group could not directly attack the 10 position to form the 10 -Oalkylamidate intermediate, and suggests that during cleavage of the nicotinamide ribosyl bond, the ribose and perhaps the acetyl group reorient to position the carbonyl oxygen for 10 attack. Further structural studies with better intermediate mimics will be required to resolve these structural discrepancies. As discussed above, productive NAD+ binding is mediated upon acetyl-substrate binding where the nicotinamide ring of NAD+ is forced into the buried site C [46,47], promoting cleavage of the ribosylnicotinamide bond through induced strain and protecting the resulting reactive intermediate (O-alkylamidate) from non-specific hydrolysis. Attack of the acetyl group from the a-face of ribose must occur to position the intermediate for efficient transfer from 10 to 20 to yield 20 -OAADPr (Figure 2). The acetyl-peptide binding specificity among sirtuins remains unclear. From the available peptide-bound strucwww.sciencedirect.com
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Figure 3
Potential pathways for the observed sirtuin-dependent ADP-ribosyltransfer to acceptor proteins.
tures [43,47,54], sirtuins appear to make contact mainly through the peptide backbone of substrate, suggesting a general lack of side-chain recognition. Consistent with this idea, SIRT1 was reported to have no substrate specificity when screened against a combinatorial acetyl-peptide library [55]. However, a steady-state kinetic analysis using a variety of mono-acetylated histone peptides as substrates revealed that in the absence of accessory proteins, ySir2, HST2 and SIRT2 exhibited varying catalytic efficiencies and displayed individual preferences for the location of the acetyl group within a given amino-acid sequence [42]. Further investigations are necessary to determine whether particular sirtuins harbor innate substrate preferences or whether targeting and other associated proteins direct specificity. Analysis of the co-enzyme specificity of sirtuins revealed an exquisite preference for NAD+. Both bases (adenine and nicotinamide) are required for tight binding, as shown by the mononucleotides NMN and www.sciencedirect.com
NAMN, which display negligible binding and do not promote deacetylation to any measurable extent. Although alterations to the adenine base appear to be tolerated [56], the nicotinamide binding pocket (site C) exhibits stringent specificity for the nicotinamide base [45,49,50,56]. Interestingly, the acid form of nicotinamide, nicotinic acid, displays negligible binding and inhibition of sirtuins even at high millimolar levels. Structural work indicates a negatively charged aspartate residue in the nicotinamide pocket that interacts with the carboxamide portion of nicotinamide [45,47]. By contrast, nicotinic acid would create highly unfavorable charge repulsion between this aspartate and disfavor binding. Avalos et al. substituted this aspartate for asparagine, and created a mutant enzyme that could now accept nicotinic acid adenine dinucleotide (NAAD) as a co-enzyme [45]. Compared with native enzyme, the mutant protein was less sensitive to nicotinamide inhibition but was more sensitive to inhibition by nicotinic acid. Current Opinion in Chemical Biology 2005, 9:431–440
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Figure 4
bitor of the forward deacetylation reaction. Consistent with a single nicotinamide binding pocket [49,50], recent structural evidence revealed free nicotinamide bound in site C [45], the same site which binds the nicotinamide of NAD+ (Figure 4). As discussed above, inhibition of deacetylation by nicotinamide involves tight binding, probably at the C site, and the subsequent attack of nicotinamide on the O-alkylamidate intermediate to regenerate NAD+ and acetylated peptide [49,50]. If a small compound could compete with free nicotinamide binding, but not react appreciably with the enzyme intermediate, it would be predicted to have a stimulatory effect on the overall deacetylation rate in the presence of nicotinamide. Recently, Sauve et al. found that the nicotinamide analog isonicotinamide (Figure 5) fulfilled the above criteria [59]. However, because high millimolar concentrations of isonicotinamide were required to observe a significant effect [59], future improvements in this strategy will be necessary to provide higher affinity and specificity.
Structural model of Sir2 with bound NAD+ and an acetylated peptide. NAD+ is shown bound in a productive conformation to Sir2Af2 with the nicotinamide ring positioned in the conserved C pocket [46]. The acetylated-lysine peptide substrate is modeled using structural alignment to the crystal structure of a p53 peptide bound to Sir2Af2 [43]. The four cysteine residues chelating the zinc (red ball) are also depicted. Atom colors: carbon (grey), oxygen (red), nitrogen (blue), sulfur (yellow), and phosphorus (orange). This figure was generated using Swiss Pdb Viewer v3.7 and POV-Ray v3.6.
Small-molecule effectors (activators and inhibitors) Over the past several years, several small-molecule activators and inhibitors of sirtuins have been reported (Figure 5). Nicotinamide, a direct product of Sir2 deacetylation, was demonstrated to be a potent physiological inhibitor of Sir2 enzymes [34,49,50,56–58]. In vivo, nicotinamide decreases gene silencing, increases rDNA recombination, and accelerates ageing in yeast, mimicking a ySir2 genetic deletion [58]. In vitro analyses indicate IC50 values in the low micromolar range with several Sir2 homologues [56]. Because nuclear nicotinamide levels are estimated to be 10–150 mM [59], it is likely that nicotinamide regulates Sir2 activity in vivo. Although nicotinamide was suggested to bind an allosteric site [58], more recent data indicates that inhibition arises from nicotinamide’s ability to condense with the high-energy enzyme:ADP ribose:acetyl-lysine intermediate to reverse the reaction, reforming NAD+ [49,50]. In doing so, nicotinamide acts as a classical non-competitive product inhiCurrent Opinion in Chemical Biology 2005, 9:431–440
A small-molecule screen for activators of human SIRT1 yielded several plant polyphenols including resveratrol, fisetin and butein (Figure 5) [60]. Resveratrol exhibited the highest activation on SIRT1 by lowering the Km for the acetylated substrate, without affecting the overall turnover rate of the enzyme [60]. Resveratrol is found in wine and is thought to harbor cardioprotective, chemopreventive and neuroprotective health benefits. Using three model organisms, resveratrol treatment increased lifespan through a Sir2-dependent pathway [60,61]. In a conflicting report, no significant increase in lifespan, telomeric silencing, or rDNA recombination was observed upon resveratrol treatment using three different yeast strains [62]. Nevertheless, that the health benefits of resveratrol were mediated through sirtuins was an intriguing possibility. The molecular basis for Sir2 activation by resveratrol has been investigated [62,63]. These in vitro studies revealed that SIRT1 activation by resveratrol was only observed when using an artificial, fluorescent acetyl-peptide as substrate (Figure 5), like the commercially available Fluor de Lys assay kit from BIOMOL (http://www.biomol.com/). Resveratrol-dependent activation was completely dependent on the presence of a covalently attached fluorophore from the artificial substrate. No resveratrol activation was observed when the same peptide, lacking the fluorophore, was employed. Biochemical and modeling studies suggest that resveratrol binding to SIRT1 promotes a conformational change that better accommodates the attached coumarin group of the artificial substrate p53-AMC. Given these recent observations, the use of resveratrol to activate SIRT1 within mammalian cells must be carefully interpreted. If indeed resveratrol acts directly on SIRT1 in vivo, one could imagine that the conformational change induced by resveratrol could lead to tighter binding and more effiwww.sciencedirect.com
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Figure 5
Small-molecule effectors of sirtuins. (a) Small molecule activators, (b) substrate, and (c) inhibitors of sirtuins.
cient turnover of native acetylated proteins. Testing resveratrol’s effect on SIRT1 with native acetylated proteins would provide evidence for this possibility. Using forward chemical genetics, several sirtuin inhibitors have been identified. Grozinger et al. screened a 1600compound library for inhibition on ySir2-mediated silencing at the telomere [64]. Sirtinol (Figure 5), a compound containing a 2-hydroxy-1-napthaldehyde moiety, was the most potent inhibitor, displaying low micromolar IC50 values against ySir2 (68 mM) and SIRT2 (38 mM). Using an analogous strategy, Bedalov et al. discovered a new class of Sir2 inhibitors [65]. These included splitomicin (Figure 5), a compound that diminished gene silencing at all three yeast silent loci and that in vitro, inhibited ySir2 with an IC50 value of 60 mM. The authors proposed that splitomicin inhibits ySir2 by competing for acetylated substrate binding. Subsequent analysis of 130 splitomicin analogues indicated the requirement for an intact lactone ring, while the naphthalene ring was dispensable for efficient ySir2 inhibition [66]. Hirao et al. identified splitomicin analogues that displayed selective inhibition of yeast Hst1 and ySir2 [67]. Tervo et al. identified 15 compounds that passed an in silico intestinal absorption test. Examination of these compounds in vitro yielded two that exhibited IC50 values in the low micromolar range [68]. Although sirtinol and splitomicin have been used as general sirtuin www.sciencedirect.com
inhibitors in mammalian cells [21,38,69], the potency and specificity of these inhibitors against mammalian sirtuins have not been evaluated. In addition, their mechanism of inhibition is unknown. Further studies evaluating the mechanism of inhibition are needed to yield rational improvements in the efficacy and specificity of these compounds.
Conclusions Recent progress in probing the molecular mechanisms for the Sir2-family of deacetylases has revealed a highly conserved structural core and catalytic mechanism. These studies support a two-step chemical mechanism involving the initial formation of a 10 -O-alkylamidate adduct formed between the acetyl-group and the nicotinamide ribose of NAD+. Subsequent attack of the ribose 20 -OH on the intermediate and addition of water yield deacetylated peptide and 20 -O-acetyl-ADP-ribose, a potential second messenger. By capturing the 10 -O-alkylamidate intermediate and driving the reaction back to reactants, the reaction product nicotinamide is a potent inhibitor both in vivo and in vitro. Small-molecule screening assays for sirtuin effectors have identified both activators (resveratrol) and inhibitors (sirtinol and splitomicin). Although much knowledge has been amassed, considerable work remains. The mechanism of inhibition by the Current Opinion in Chemical Biology 2005, 9:431–440
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identified small-molecules has not been determined. Improvements to the development of more specific and tighter-binding effector molecules will be necessary before their potential use as therapeutics. The physiological link between sirtuin function and the NAD+-dependence and the formation the metabolite OAADPr remains elusive. Further studies are needed to provide unbiased screening of potential acetylated targets of sirtuins. The potential function of sirtuins as ADP-ribosyltransferases has not been adequately addressed at either the molecular or cellular level.
Acknowledgements I thank laboratory members Margie Borra and Brian Smith for their critical reading of the manuscript. I thank Brian Smith for the structural modeling and construction of Figure 4, as well as for his assistance with Figure 5. JMD was supported by NIH Grant GM065386.
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26. Tanner KG, Landry J, Sternglanz R, Denu JM: Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADPribose. Proc Natl Acad Sci USA 2000, 97:14178-14182. 27. Tanny JC, Moazed D: Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product. Proc Natl Acad Sci USA 2001, 98:415-420. 28. Imai S, Armstrong CM, Kaeberlein M, Guarente L: Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 2000, 403:795-800. 29. Sauve AA, Celic I, Avalos J, Deng H, Boeke JD, Schramm VL: Chemistry of gene silencing: the mechanism of NAD+dependent deacetylation reactions. Biochemistry 2001, 40:15456-15463. 30. Jackson MD, Denu JM: Structural identification of 2(and 30 -O-acetyl-ADP-ribose as novel metabolites derived from www.sciencedirect.com
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the Sir2 family of beta-NAD+-dependent histone/protein deacetylases. J Biol Chem 2002, 277:18535-18544. 31. Chang JH, Kim HC, Hwang KY, Lee JW, Jackson SP, Bell SD, Cho Y: Structural basis for the NAD-dependent deacetylase mechanism of Sir2. J Biol Chem 2002, 277:34489-34498. 32. Grozinger CM, Schreiber SL: Deacetylase enzymes: biological functions and the use of small-molecule inhibitors. Chem Biol 2002, 9:3-16. 33. Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Cohen H, Lin SS, Manchester JK, Gordon JI, Sinclair DA: Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J Biol Chem 2002, 277:18881-18890. This study along with [34,35,36] established a connection between enzymes of the yeast NAD+ salvage pathway and Sir2 functions in gene silencing and ageing. 34. Gallo CM, Smith DL Jr, Smith JS: Nicotinamide clearance by Pnc1 directly regulates Sir2-mediated silencing and longevity. Mol Cell Biol 2004, 24:1301-1312. 35. Sandmeier JJ, Celic I, Boeke JD, Smith JS: Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway. Genetics 2002, 160:877-889. 36. Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Sinclair DA: Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 2003, 423:181-185. The authors reported evidence that nicotinamide is a cellular inhibitor of ySir2 and that PNC1, a nicotinamidase of the NAD salvage pathway, controls cellular nicotinamide levels and Sir2 activity. 37. Revollo JR, Grimm AA, Imai S: The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J Biol Chem 2004, 279:50754-50763. This study, along with [38], demonstrated that NAD biosynthetic enzymes can regulate SIRT1 function in mammalian cells. 38. Araki T, Sasaki Y, Milbrandt J: Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 2004, 305:1010-1013. In addition to indicating that nuclear NAD biosynthesis can upregulate SIRT1, this work suggested that SIRT1 functions in protecting neuronal cells from injury such as oxidant insult. 39. Denu JM: Linking chromatin function with metabolic networks: Sir2 family of NAD(+)-dependent deacetylases. Trends Biochem Sci 2003, 28:41-48. 40. Borra MT, O’Neill FJ, Jackson MD, Marshall B, Verdin E, Foltz KR, Denu JM: Conserved enzymatic production and biological effect of O-acetyl-ADP-ribose by silent information regulator 2-like NAD+-dependent deacetylases. J Biol Chem 2002, 277:12632-12641. The production of O-acetyl-ADP-ribose by sirtuins was shown to be universally conserved among active enzymes from diverse species. Also, this was the first report to show the biological effects of O-acetyl-ADPribose by microinjection.
46. Avalos JL, Boeke JD, Wolberger C: Structural basis for the mechanism and regulation of Sir2 enzymes. Mol Cell 2004, 13:639-648. This study was the first to establish the complete NAD+ binding surface, highlighted by nicotinamide bound to the previously described C pocket [44]. 47. Zhao K, Harshaw R, Chai X, Marmorstein R: Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD(+)dependent Sir2 histone/protein deacetylases. Proc Natl Acad Sci USA 2004, 101:8563-8568. A ternary complex between HST2, a histone acetyl-peptide and an NAD analog provided additional structural evidence for the mechanism of catalysis by sirtuins. 48. Sauve AA, Schramm VL: SIR2: the biochemical mechanism of NAD(+)-dependent protein deacetylation and ADP-ribosyl enzyme intermediates. Curr Med Chem 2004, 11:807-826. 49. Jackson MD, Schmidt MT, Oppenheimer NJ, Denu JM: Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases. J Biol Chem 2003, 278:50985-50998. This study provided the molecular insight into the mechanisms of catalysis and of nicotinamide inhibition by sirtuins. 50. Sauve AA, Schramm VL: Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry. Biochemistry 2003, 42:9249-9256. This study describes the mechanistic basis for nicotinamide inhibition, and suggested the use of nicotinamide analogs as sirtuin activators. 51. Tanny JC, Dowd GJ, Huang J, Hilz H, Moazed D: An enzymatic activity in the yeast Sir2 protein that is essential for gene silencing. Cell 1999, 99:735-745. 52. Garcia-Salcedo JA, Gijon P, Nolan DP, Tebabi P, Pays E: A chromosomal SIR2 homologue with both histone NADdependent ADP-ribosyltransferase and deacetylase activities is involved in DNA repair in Trypanosoma brucei. EMBO J 2003, 22:5851-5862. 53. Liszt G, Ford E, Kurtev M, Guarente L: Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J Biol Chem 2005, 280:21313-21320. 54. Zhao K, Chai X, Marmorstein R: Structure of the yeast Hst2 protein deacetylase in ternary complex with 20 -O-acetyl ADP ribose and histone peptide. Structure (Camb) 2003, 11:1403-1411. 55. Blander G, Olejnik J, Krzymanska-Olejnik E, McDonagh T, Haigis M, Yaffe MB, Guarente L: SIRT1 shows no substrate specificity in vitro. J Biol Chem 2005, 280:9780-9785. 56. Schmidt MT, Smith BC, Jackson MD, Denu JM: Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation. J Biol Chem 2004, 279:40122-40129. 57. Landry J, Slama JT, Sternglanz R: Role of NAD(+) in the deacetylase activity of the SIR2-like proteins. Biochem Biophys Res Commun 2000, 278:685-690.
41. Rafty LA, Schmidt MT, Perraud AL, Scharenberg AM, Denu JM: Analysis of O-acetyl-ADP-ribose as a target for Nudix ADP-ribose hydrolases. J Biol Chem 2002, 277:47114-47122.
58. Bitterman KJ, Anderson RM, Cohen HY, Latorre-Esteves M, Sinclair DA: Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem 2002, 277:45099-45107.
42. Borra MT, Langer MR, Slama JT, Denu JM: Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases. Biochemistry 2004, 43:9877-9887. This kinetic study established the complete kinetic mechanism for sirtuin enzymes. Acetyl-peptide substrate analysis provided evidence that the catalytic domains of sirtuins exhibit substrate preferences.
59. Sauve AA, Moir RD, Schramm VL, Willis IM: Chemical activation of sir2- dependent silencing by relief of nicotinamide inhibition. Mol Cell 2005, 17:595-601. These authors used isonicotinamide as an activator of ySir2 in vitro and in vivo. This work suggests the possibility of developing sirtuin activators that compete for the nicotinamide binding site and relieve endogenous nicotinamide inhibition.
43. Avalos JL, Celic I, Muhammad S, Cosgrove MS, Boeke JD, Wolberger C: Structure of a Sir2 enzyme bound to an acetylated p53 peptide. Mol Cell 2002, 10:523-535. This binary complex structure was the first to establish the acetyl-peptide binding site within sirtuins.
60. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL et al.: Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003, 425:191-196.
44. Min J, Landry J, Sternglanz R, Xu RM: Crystal structure of a SIR2 homolog-NAD complex. Cell 2001, 105:269-279. 45. Avalos JL, Bever KM, Wolberger C: Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol Cell 2005, 17:855-868. www.sciencedirect.com
61. Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D: Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature 2004, 430:686-689. The authors of this study and that of [60] report the anti-aging effects of resveratrol and suggest that Sir2 proteins are the direct in vivo targets of resveratrol, leading to activation of protein deacetylation. Current Opinion in Chemical Biology 2005, 9:431–440
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62. Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, Caldwell S, Napper A, Curtis R, Distefano PS, Fields S et al.: Substrate specific activation of sirtuins by resveratrol. J Biol Chem 2005, 280:17038-17045. This study along with that of [63] demonstrated that the apparent in vitro activation of sirtuins by resveratrol results from the use of artificial fluorescent peptides. In yeast, these authors report no Sir2 activation by resveratrol. 63. Borra MT, Smith BC, Denu JM: Mechanism of human SIRT1 activation by resveratrol. J Biol Chem 2005, 280:17187-17195. This report provided the mechanistic basis for the apparent activation of SIRT1 by resveratrol, when a variety of both native and fluorescently labeled peptides are employed as substrates. 64. Grozinger CM, Chao ED, Blackwell HE, Moazed D, Schreiber SL: Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 2001, 276:38837-38843. Using a chemical genetics approach, these authors identified the molecule sirtinol as an inhibitor of ySir2.
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65. Bedalov A, Gatbonton T, Irvine WP, Gottschling DE, Simon JA: Identification of a small molecule inhibitor of Sir2p. Proc Natl Acad Sci USA 2001, 98:15113-15118. Using a chemical genetics approach, splitomicin was discovered as an inhibitor of sirtuins. 66. Posakony J, Hirao M, Stevens S, Simon JA, Bedalov A: Inhibitors of Sir2: evaluation of splitomicin analogues. J Med Chem 2004, 47:2635-2644. 67. Hirao M, Posakony J, Nelson M, Hruby H, Jung M, Simon JA, Bedalov A: Identification of selective inhibitors of NAD+dependent deacetylases using phenotypic screens in yeast. J Biol Chem 2003, 278:52773-52782. 68. Tervo AJ, Kyrylenko S, Niskanen P, Salminen A, Leppanen J, Nyronen TH, Jarvinen T, Poso A: An in silico approach to discovering novel inhibitors of human sirtuin type 2. J Med Chem 2004, 47:6292-6298. 69. Fulco M, Schiltz RL, Iezzi S, King MT, Zhao P, Kashiwaya Y, Hoffman E, Veech RL, Sartorelli V: Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state. Mol Cell 2003, 12:51-62.
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Current Opinion in Chemical Biology 2005, 9:431–440 This review comes from a themed issue on Mechanisms Edited by Rowena G Matthews and Christopher T Walsh Available online 24th August 2005 1367-5931/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2005.08.010
Introduction The NAD+-dependent protein deacetylases have emerged as important regulators of diverse biological processes [1,2]. These enzymes, referred to as sirtuins or Sir2-like proteins, constitute the class III histone deacetylases (HDACs) and are conserved from bacteria to humans [3,4]. The founding member yeast Sir2 (ySir2, yeast silent information regulator 2) is essential for maintaining silent chromatin through the deacetylation of histones (Figure 1a) [5,6]. Sir2 enzymes have also been implicated in mediating lifespan increases in yeast, worms and flies [7–9]. Additional reports over the past several years indicate a much broader range of substrates and functions for sirtuins [1,10,11]. For example, short-chain fatty acyl-coenzyme A (adenosine monophosphate-forming) synthetases in bacteria and yeast are regulated by reversible acetylation, with www.sciencedirect.com
sirtuins functioning as activating deacetylases [12,13]. Among the most studied human Sir2 homologs, SIRT1 was shown to regulate non-histone substrates including p53 [14–16], FOXO proteins [17–19], p300 [20], NFkB [21] and PGC-1a [22,23] (Figure 1b), implicating sirtuins in apoptosis, cell survival, transcription and metabolism. The role of sirtuins in controlling basic cellular functions, particularly apoptosis and cell survival, has spurred great interest in understanding the molecular mechanisms of catalysis, substrate specificity and regulation, as well as in the development of small-molecule regulators that either activate or inhibit their cellular function. Pharmaceuticals that target sirtuins may be useful in the treatment of ageing, cancer, diabetes and neurodegeneration. Here, recent progress in our knowledge of catalysis, structure, regulation, substrate specificity and small-molecule effectors is presented.
Deacetylation reaction and biological implications In cases where clear enzymatic activity has been measured, sirtuins catalyze NAD+-dependent e–N-acetyl-lysine deacetylation from histones and non-histone proteins [24–28]. The deacetylase reaction requires NAD+ and produces deacetylated protein, nicotinamide and the unique metabolite O-acetyl-ADP-ribose (OAADPr) (Figure 1b) [26,27,29]. Sirtuins transfer the acetyl group from proteins and peptides to the 20 -OH of the nicotinamide ribose, yielding 20 -O-acetyl-ADP-ribose (20 -OAADPr) [29–31]. The nicotinamide ribosyl bond is cleaved and one net water molecule is added to the nicotinamide ribose. This reaction is in stark contrast to class I and II histone/protein deacetylases, which utilize an active site zinc, produce acetate, and do not require NAD+ [32]. The uniqueness of the sirtuin reaction begs many fundamental questions concerning the catalytic mechanism and its biological relevance. Why is NAD+ consumption required for a deacetylation reaction that is already thermodynamically favored? And, why is the acetyl group specifically transferred not to water but to the ADP-ribose portion of NAD+ to generate OAADPr? How does the enzyme accomplish this novel chemical reaction? Coupling deacetylation to NAD+ would be one mechanism to link changes in cellular NAD+ levels with deacetylation activity. Although a strong correlation between total NAD+ levels in cells and Sir2 function remains to be established, several genetic studies have provided ample evidence for a link between enzymes involved in NAD+ biosynthesis and sirtuin function [33,34,35,36–38]. It may be that localized synthesis of NAD+ is directly responsible for enhanced ySir2 and SIRT1 activity in these studies. Another explanation for Current Opinion in Chemical Biology 2005, 9:431–440
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Figure 1
Proposed functions of yeast Sir2 and of mammalian SIRT1. (a) Yeast Sir2 histone deacetylation and silencing function. Acetyl-CoA-dependent histone acetyltransferases acetylate histones leading to transcriptional activation. The NAD+-dependent deacetylase Sir2 deacetylates histones and creates transcriptional silent chromatin. Nicotinamide is a product of the reaction and is a potent inhibitor of Sir2. OAADPr is a novel metabolite generated during deacetylation and may function in linked cellular pathways. An NAD+ salvage pathway depletes nicotinamide and funnels NAD+ to Sir2. (b) General chemical reaction catalyzed by sirtuins. Human SIRT1 is depicted with several reported acetylated protein targets and the pathways affected.
the NAD+ dependence is that protein deacetylation is designed to create OAADPr, a proposed second messenger [39]. Microinjection of OAADPr was shown to inhibit oocyte maturation and to block cell division in starfish blastomeres [40]. Unidentified enzymes found in several species including human cells are able to efficiently metabolize OAADPr in extracts [40,41], suggesting that cellular pathways exist to tightly control OAADPr levels. Thus, OAADPr may act as a secondary messenger, a cofactor, or as a metabolic intermediate that couples deacetylation of target proteins to other cellular pathways [39]. Further studies are needed to uncover the functions of OAADPr, particularly its role in known processes regulated by sirtuins. Current Opinion in Chemical Biology 2005, 9:431–440
Reaction mechanism Recent kinetic studies indicate that sirtuins first bind acetylated substrate followed by NAD+ to form a ternary enzyme complex [42]. Conformational rearrangement of the NAD+ binding pocket upon acetyl-peptide binding was observed in the X-ray structure of a binary complex between archaeal Sir2 enzyme, Sir2-Af2, and an acetylated p53 peptide [43], providing additional evidence for ordered substrate binding. Although binary complexes of enzyme with NAD+ or ADP-ribose were reported initially [31,44], the nicotinamide moiety was not observed until recently, when the position of the nicotinamide base was pinpointed within the highly conserved C-pocket [45,46,47], a buried depression previously www.sciencedirect.com
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Figure 2
Proposed catalytic mechanism for the sir2 family of deacetylases.
noted near the predicted active site and implicated in catalysis [44]. As proposed in Figure 2, the initial chemical step involves cleavage of the nicotinamide-ribosyl bond of NAD+ and the concomitant attack of the carbonyl oxygen of an acetyl group from bound peptide to form a 10 -O-alkylamidate adduct between the two substrates (reviewed in [39,48]). Nicotinamide is released and subsequent activation of the ribose 20 -OH leads to nucleophilic attack at the iminium intermediate and generation of a 10 -20 -cyclic species. Water attack on the cyclic intermediate and general-acid catalyzed elimination of the e-amine of lysine yields the final two products 20 -OAADPr and deacetylated peptide, which are released in random fashion [42]. Evidence for a two-step chemical mechanism comes from the observation of enzyme-catalyzed nicotinamide:NAD+ www.sciencedirect.com
exchange (transglycosidation) [24,49,50], from rapidquench flow studies [26,42] and from the use of 20 deoxy-20 -fluoro-NAD+ as a surrogate co-substrate [49]. In the presence of acetyl-peptide and NAD+, exogenously added [14C]-nicotinamide is quickly incorporated into [14C]-NAD+ during the nicotinamide:NAD+ exchange reaction. This rapid transglycosidation with free nicotinamide to regenerate b-NAD+ is proposed to occur by the facile nucleophilic attack of nicotinamide on the putative 10 -O-alkylamidate intermediate [49,50]. Consistent with the formation of a 10 -O-alkylamidate adduct during deacetylation, replacement of NAD+ with 20 -deoxy-20 fluoro-NAD+ halted the deacetylation reaction but not the nicotinamide exchange reaction [49]. Rapid quenching studies with HST2 demonstrated that the nicotinamide–ribosyl bond is cleaved to form the putative 10 -O-alkylamidate intermediate at a rate of 8 s1, while the rate of subsequent acetyl-transfer to the 20 -OH ribose Current Opinion in Chemical Biology 2005, 9:431–440
434 Mechanisms
occurred at a rate of 2 s1. The kcat is 0.2 s1, suggesting that a product release step may limit turnover [42]. Sirtuin deacetylation reactions carried out in 18OH2, incorporate one 18O atom into the acetyl group of product 20 -OAADPr ([29]; JM Denu, BC Smith, unpublished data). Recently, the fate of the carbonyl oxygen of the acetylated peptide was determined using an 18O-label in the acetyl group of substrate (JM Denu, BC Smith, unpublished data). These recent studies demonstrated that the carbonyl oxygen of substrate is enzymatically transferred to the 10 position of product 20 -OAADPr. These new data provide the first direct evidence that the acetyl-group carbonyl oxygen initially attacks at the 10 position to yield the proposed 10 -O-alkylamidate intermediate (Figure 2). In the last chemical step, solvent isotope exchange data ([29]; JM Denu, BC Smith, unpublished data) are consistent with a water molecule attacking the 10 -20 -cyclic intermediate, incorporating one solvent molecule into the acetyl group of 20 -OAADPr. Crystal data have shown that the side chain of an invariant histidine residue is hydrogen bonded to the 30 -OH of the nicotinamide ribose and is suggested to act as a general base by initiating the deprotonation of the 20 -OH (acting through the 30 -OH) for attack on the 10 -O-alkylamidate intermediate, forming the 10 -20 -cyclic intermediate (Figure 2). This same histidine residue may serve as a general acid in the last step of catalysis, protonating the lysine leaving group (Figure 2).
Adp-ribosylation In several reports, sirtuins were suggested to harbor NAD+-dependent ADP-ribosyltransferase activity, transferring ADP-ribose to a protein acceptor or to the enzyme itself [3,28,51–53]. Weak or nonexistent deacetylation activity from some sirtuins has fueled this notion. However, there is currently insufficient evidence to unequivocally assign bona fide protein ADP-ribosyltransferase activity. Although sirtuins acting as ADP-ribosyltransferases remains a distinct possibility, the ability of sirtuins to catalyze multiple rounds of protein ADP-ribosylation has not been demonstrated. Among the reported cases, there are three possible mechanisms for the observed ADP-ribosylation (Figure 3). The first possibility is that some sirtuins indeed do possess ADP-ribosyltransferase activity, transferring the ADP-ribose of NAD+ to an acceptor protein (Figure 3a). In this scenario, an acetylated substrate is not required. In a second possibility, an acetylated substrate and NAD+ would be required to initiate the formation of the O-alkylamidate intermediate, which may be susceptible to directed or non-specific nucleophilic attack by the acceptor protein (Figure 3b), which may include acetylated protein itself. This mechanism is similar to the reaction (transglycosidation) of nicotinamide with the enzyme intermediate. After collapse of the intermediate to form the ADP-ribosylated Current Opinion in Chemical Biology 2005, 9:431–440
product, the acetylated residue would be regenerated. A third model requires NAD+ and acetylated substrate to produce OAADPr or its hydrolyzed product ADP-ribose (Figure 3c). Both OAADPr and ADP-ribose can react nonenzymatically with proteins to yield the ADP-ribosylated protein products (T Kowieski, JM Denu, unpublished data). The observation of sirtuin-dependent ADP-ribosylation with hemi-acetylated histone preparations should be analyzed with caution, as the resulting OAADPr produced by deacetylation can react with resident proteins non-enzymatically. In vivo, the possibility that OAADPr could serve as a substrate for a novel ADPribosyltransferase should be considered as well.
Structure and substrate specificity Recent X-ray structures of liganded sirtuin complexes have defined the binding pockets for both substrates and have provided supporting evidence for the mechanism proposed in Figure 2. The catalytic core of sirtuins consists of an NAD+-binding domain that is a variant of the Rossmann fold and a smaller subdomain composed of a helical module and a zinc-binding module (Figure 4). A large groove at the interface creates the active site where NAD+ binds. Acetylated peptide binds along an enzyme cleft and forms an enzyme-substrate b sheet with two flanking strands from the enzyme [43]. The acetyl-lysine side chain protrudes into a hydrophobic tunnel that terminates near the nicotinamide ribose of NAD+ [46,47]. Modeling studies based on the binary complexes of Sir2Af2 with NAD+ [46] and of Sir2Af2 with an acetylated-lysine peptide [43] suggest that the acetyl-group would be positioned on the a-face of the nicotinamide ribose of the catalytically competent ternary complex (Figure 4). Zhao et al. reported the ternary structure of HST2 bound to both a non-hydrolyzable NAD+ analog and an acetylate histone peptide [47]. These authors observed the acetyl group hydrogenbonding to the ribose 20 and 30 OH of the NAD+ analog. In this orientation, the carbonyl oxygen of the acetyl-group could not directly attack the 10 position to form the 10 -Oalkylamidate intermediate, and suggests that during cleavage of the nicotinamide ribosyl bond, the ribose and perhaps the acetyl group reorient to position the carbonyl oxygen for 10 attack. Further structural studies with better intermediate mimics will be required to resolve these structural discrepancies. As discussed above, productive NAD+ binding is mediated upon acetyl-substrate binding where the nicotinamide ring of NAD+ is forced into the buried site C [46,47], promoting cleavage of the ribosylnicotinamide bond through induced strain and protecting the resulting reactive intermediate (O-alkylamidate) from non-specific hydrolysis. Attack of the acetyl group from the a-face of ribose must occur to position the intermediate for efficient transfer from 10 to 20 to yield 20 -OAADPr (Figure 2). The acetyl-peptide binding specificity among sirtuins remains unclear. From the available peptide-bound strucwww.sciencedirect.com
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Figure 3
Potential pathways for the observed sirtuin-dependent ADP-ribosyltransfer to acceptor proteins.
tures [43,47,54], sirtuins appear to make contact mainly through the peptide backbone of substrate, suggesting a general lack of side-chain recognition. Consistent with this idea, SIRT1 was reported to have no substrate specificity when screened against a combinatorial acetyl-peptide library [55]. However, a steady-state kinetic analysis using a variety of mono-acetylated histone peptides as substrates revealed that in the absence of accessory proteins, ySir2, HST2 and SIRT2 exhibited varying catalytic efficiencies and displayed individual preferences for the location of the acetyl group within a given amino-acid sequence [42]. Further investigations are necessary to determine whether particular sirtuins harbor innate substrate preferences or whether targeting and other associated proteins direct specificity. Analysis of the co-enzyme specificity of sirtuins revealed an exquisite preference for NAD+. Both bases (adenine and nicotinamide) are required for tight binding, as shown by the mononucleotides NMN and www.sciencedirect.com
NAMN, which display negligible binding and do not promote deacetylation to any measurable extent. Although alterations to the adenine base appear to be tolerated [56], the nicotinamide binding pocket (site C) exhibits stringent specificity for the nicotinamide base [45,49,50,56]. Interestingly, the acid form of nicotinamide, nicotinic acid, displays negligible binding and inhibition of sirtuins even at high millimolar levels. Structural work indicates a negatively charged aspartate residue in the nicotinamide pocket that interacts with the carboxamide portion of nicotinamide [45,47]. By contrast, nicotinic acid would create highly unfavorable charge repulsion between this aspartate and disfavor binding. Avalos et al. substituted this aspartate for asparagine, and created a mutant enzyme that could now accept nicotinic acid adenine dinucleotide (NAAD) as a co-enzyme [45]. Compared with native enzyme, the mutant protein was less sensitive to nicotinamide inhibition but was more sensitive to inhibition by nicotinic acid. Current Opinion in Chemical Biology 2005, 9:431–440
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Figure 4
bitor of the forward deacetylation reaction. Consistent with a single nicotinamide binding pocket [49,50], recent structural evidence revealed free nicotinamide bound in site C [45], the same site which binds the nicotinamide of NAD+ (Figure 4). As discussed above, inhibition of deacetylation by nicotinamide involves tight binding, probably at the C site, and the subsequent attack of nicotinamide on the O-alkylamidate intermediate to regenerate NAD+ and acetylated peptide [49,50]. If a small compound could compete with free nicotinamide binding, but not react appreciably with the enzyme intermediate, it would be predicted to have a stimulatory effect on the overall deacetylation rate in the presence of nicotinamide. Recently, Sauve et al. found that the nicotinamide analog isonicotinamide (Figure 5) fulfilled the above criteria [59]. However, because high millimolar concentrations of isonicotinamide were required to observe a significant effect [59], future improvements in this strategy will be necessary to provide higher affinity and specificity.
Structural model of Sir2 with bound NAD+ and an acetylated peptide. NAD+ is shown bound in a productive conformation to Sir2Af2 with the nicotinamide ring positioned in the conserved C pocket [46]. The acetylated-lysine peptide substrate is modeled using structural alignment to the crystal structure of a p53 peptide bound to Sir2Af2 [43]. The four cysteine residues chelating the zinc (red ball) are also depicted. Atom colors: carbon (grey), oxygen (red), nitrogen (blue), sulfur (yellow), and phosphorus (orange). This figure was generated using Swiss Pdb Viewer v3.7 and POV-Ray v3.6.
Small-molecule effectors (activators and inhibitors) Over the past several years, several small-molecule activators and inhibitors of sirtuins have been reported (Figure 5). Nicotinamide, a direct product of Sir2 deacetylation, was demonstrated to be a potent physiological inhibitor of Sir2 enzymes [34,49,50,56–58]. In vivo, nicotinamide decreases gene silencing, increases rDNA recombination, and accelerates ageing in yeast, mimicking a ySir2 genetic deletion [58]. In vitro analyses indicate IC50 values in the low micromolar range with several Sir2 homologues [56]. Because nuclear nicotinamide levels are estimated to be 10–150 mM [59], it is likely that nicotinamide regulates Sir2 activity in vivo. Although nicotinamide was suggested to bind an allosteric site [58], more recent data indicates that inhibition arises from nicotinamide’s ability to condense with the high-energy enzyme:ADP ribose:acetyl-lysine intermediate to reverse the reaction, reforming NAD+ [49,50]. In doing so, nicotinamide acts as a classical non-competitive product inhiCurrent Opinion in Chemical Biology 2005, 9:431–440
A small-molecule screen for activators of human SIRT1 yielded several plant polyphenols including resveratrol, fisetin and butein (Figure 5) [60]. Resveratrol exhibited the highest activation on SIRT1 by lowering the Km for the acetylated substrate, without affecting the overall turnover rate of the enzyme [60]. Resveratrol is found in wine and is thought to harbor cardioprotective, chemopreventive and neuroprotective health benefits. Using three model organisms, resveratrol treatment increased lifespan through a Sir2-dependent pathway [60,61]. In a conflicting report, no significant increase in lifespan, telomeric silencing, or rDNA recombination was observed upon resveratrol treatment using three different yeast strains [62]. Nevertheless, that the health benefits of resveratrol were mediated through sirtuins was an intriguing possibility. The molecular basis for Sir2 activation by resveratrol has been investigated [62,63]. These in vitro studies revealed that SIRT1 activation by resveratrol was only observed when using an artificial, fluorescent acetyl-peptide as substrate (Figure 5), like the commercially available Fluor de Lys assay kit from BIOMOL (http://www.biomol.com/). Resveratrol-dependent activation was completely dependent on the presence of a covalently attached fluorophore from the artificial substrate. No resveratrol activation was observed when the same peptide, lacking the fluorophore, was employed. Biochemical and modeling studies suggest that resveratrol binding to SIRT1 promotes a conformational change that better accommodates the attached coumarin group of the artificial substrate p53-AMC. Given these recent observations, the use of resveratrol to activate SIRT1 within mammalian cells must be carefully interpreted. If indeed resveratrol acts directly on SIRT1 in vivo, one could imagine that the conformational change induced by resveratrol could lead to tighter binding and more effiwww.sciencedirect.com
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Figure 5
Small-molecule effectors of sirtuins. (a) Small molecule activators, (b) substrate, and (c) inhibitors of sirtuins.
cient turnover of native acetylated proteins. Testing resveratrol’s effect on SIRT1 with native acetylated proteins would provide evidence for this possibility. Using forward chemical genetics, several sirtuin inhibitors have been identified. Grozinger et al. screened a 1600compound library for inhibition on ySir2-mediated silencing at the telomere [64]. Sirtinol (Figure 5), a compound containing a 2-hydroxy-1-napthaldehyde moiety, was the most potent inhibitor, displaying low micromolar IC50 values against ySir2 (68 mM) and SIRT2 (38 mM). Using an analogous strategy, Bedalov et al. discovered a new class of Sir2 inhibitors [65]. These included splitomicin (Figure 5), a compound that diminished gene silencing at all three yeast silent loci and that in vitro, inhibited ySir2 with an IC50 value of 60 mM. The authors proposed that splitomicin inhibits ySir2 by competing for acetylated substrate binding. Subsequent analysis of 130 splitomicin analogues indicated the requirement for an intact lactone ring, while the naphthalene ring was dispensable for efficient ySir2 inhibition [66]. Hirao et al. identified splitomicin analogues that displayed selective inhibition of yeast Hst1 and ySir2 [67]. Tervo et al. identified 15 compounds that passed an in silico intestinal absorption test. Examination of these compounds in vitro yielded two that exhibited IC50 values in the low micromolar range [68]. Although sirtinol and splitomicin have been used as general sirtuin www.sciencedirect.com
inhibitors in mammalian cells [21,38,69], the potency and specificity of these inhibitors against mammalian sirtuins have not been evaluated. In addition, their mechanism of inhibition is unknown. Further studies evaluating the mechanism of inhibition are needed to yield rational improvements in the efficacy and specificity of these compounds.
Conclusions Recent progress in probing the molecular mechanisms for the Sir2-family of deacetylases has revealed a highly conserved structural core and catalytic mechanism. These studies support a two-step chemical mechanism involving the initial formation of a 10 -O-alkylamidate adduct formed between the acetyl-group and the nicotinamide ribose of NAD+. Subsequent attack of the ribose 20 -OH on the intermediate and addition of water yield deacetylated peptide and 20 -O-acetyl-ADP-ribose, a potential second messenger. By capturing the 10 -O-alkylamidate intermediate and driving the reaction back to reactants, the reaction product nicotinamide is a potent inhibitor both in vivo and in vitro. Small-molecule screening assays for sirtuin effectors have identified both activators (resveratrol) and inhibitors (sirtinol and splitomicin). Although much knowledge has been amassed, considerable work remains. The mechanism of inhibition by the Current Opinion in Chemical Biology 2005, 9:431–440
438 Mechanisms
identified small-molecules has not been determined. Improvements to the development of more specific and tighter-binding effector molecules will be necessary before their potential use as therapeutics. The physiological link between sirtuin function and the NAD+-dependence and the formation the metabolite OAADPr remains elusive. Further studies are needed to provide unbiased screening of potential acetylated targets of sirtuins. The potential function of sirtuins as ADP-ribosyltransferases has not been adequately addressed at either the molecular or cellular level.
Acknowledgements I thank laboratory members Margie Borra and Brian Smith for their critical reading of the manuscript. I thank Brian Smith for the structural modeling and construction of Figure 4, as well as for his assistance with Figure 5. JMD was supported by NIH Grant GM065386.
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